Culture supernatants from alloantigen- and Con A-stimulated mouse spleen cells contain T cell-derived factors which augment both humoral and cell mediated immune responses in vitro. The biochemical relationship between these factors and thymocyte mitogenic factor has been investigated. Thymocyte mitogenic factor (MW 38,000) has been found to be chromatographically inseparable, following a multiple step purification scheme, from the soluble mediator which enhances the antibody response of nude mouse spleen cells and from a mediator which restores the antigen-dependent cytotoxic response of mouse thymocytes. Thus, the data support the hypothesis that thymocyte mitogenic factor is a component in the activation pathways for both humoral (B-cell-mediated) and cellular (T-cell-mediated) immune responses. In addition, a second cytotoxic cell helper factor (MW 40,000) was similarly found to be inseparable from type II interferon. The finding of a second biochemically distinct cytotoxic cell factor (interferon) within the same supernatant illustrates the point that multiple helper factors with perhaps different mechanisms-of-action may function within the same activation pathway.